WO1990010080A1 - Production of nucleoside - Google Patents

Abstract

This invention relates to a method of producing a nucleoside by reacting a base donor, a sugar residue donor, and a phosphate donor with one another in the presence of an enzymatic preparation containing a nucleoside phosphorylase to thereby form an N-glycoside linkage between a base portion of the base donor and a sugar residue portion of the sugar residue donor, characterized in that said enzymatic preparation containing a nucleoside phosphorylase is one based on the cells of at least one microorganism selected from among those belonging to thermophilic bacteria of the genus Bacillus and having a high nucleoside phosphorylase activity per unit weight of the cell.

Description

 Technical Documents Manufacturing Method of Nucleoside Technical Field

 The present invention relates to a method for producing a nucleoside using an enzyme preparation containing a nucleoside phosphorylase derived from a thermophilic bacterium belonging to the genus Bacillus.

 Background technology

Methods for producing nucleosides by enzymatically reacting a sugar residue donor such as nucleoside, ribose-1-phosphate or the like with a base donor include, for example, methods for producing various nucleosides. (JP-B-43-24475, JP-B-43-28954, JP-B-43-28955, JP-B-43-28956, JP-B-45-111116, JP-B-48-14957, JP-A-Showa 55-714995, JP-A-56-18599, JP-A-56-142293, JP-A-56-164793, JP-A-56-166199, JP-A-58-63393, JP-A-58-94396, JP-A-58-170493, etc., and methods for producing various pyrimidine nucleosides (JP-B-35-16478, JP-A-56-102794, JP-A-Showa) 59-21331, JP-A-60-239495, JP-A-60-239495, etc.) Methods for producing nucleotides (Japanese Patent Application Laid-Open No. 50-29720, Japanese Patent Application Laid-Open No. 57-146593, Japanese Patent Application Laid-Open No. 58-190396, Japanese Patent Application No. 58-216696, Japanese Patent Application

143599, JP-A-59-179094, JP-A-59-213397, JP-A-60-1 20981, JP-A-60-133896, JP-A-63-31093, JP-A-63-1 77797).

 However, these enzymatic methods for producing nucleosides are superior to chemical synthesis methods in terms of the substrate specificity and stereoselectivity specific to enzymatic reactions, but the enzyme ability is not sufficient. However, it was not always satisfactory in terms of yield and yield.

 In addition, if the reaction is carried out at room temperature, a decrease in yield, which is considered to be caused by contamination by various bacteria, is also observed. There was also a problem that the yield was remarkably reduced as a result.

In general, the synthesis of a compound is brought about by the fact that the equilibrium between the production reaction and the decomposition reaction is inclined toward the production reaction.Therefore, in order to increase the yield and yield of the compound, the production reaction must be promoted and the decomposition It is essential to suppress the reaction, and this principle is no exception in the production of enzymatic compounds. In addition, when the reaction temperature is increased, the reaction rate is increased, the reaction is completed in a short time, and the solubility of the substrate is also increased. ing.

 When producing nucleosides using nucleoside phosphorylase, two factors must be considered in order to accelerate the nucleoside production reaction: the ability of the enzyme itself to be used as a catalyst and the reaction conditions. No. The selection of reaction conditions is only a supplementary step to bring out the ability of the enzyme to be used, and a drastic method for promoting the production reaction and increasing the yield of the target compound has excellent ability. This is to use nucleoside phosphorylase.

 Most of the conventional nucleoside phosphorylases used for the production of nucleosides are prepared from microorganisms that can be easily prepared. However, when the ability of the enzyme was examined from the viewpoints of reaction efficiency, specific activity, heat resistance, yield of the target compound, etc., those conventionally used were not always satisfactory.

On the other hand, with regard to enzymes considered to be involved in the decomposition reaction of the produced nucleosides, for example, nucleosidase, a method for suppressing nucleosidase by immobilization using a photocurable resin has been reported (Japanese Patent Application Laid-Open No. — 25 3 3 9 3), This method is an excellent method, but it is difficult to immobilize some microorganisms when using microbial cells as an enzyme preparation. Some methods were not so versatile.

 The present inventors screened various microorganisms to find an enzyme having excellent ability to be used for enzymatic production of nucleosides. We discovered a group of microorganisms that contain high levels of nucleoside phosphorylase with high activity and thermostability and high nucleoside phosphorylase activity per unit weight of cells.

 Heretofore, nucleoside dofoliphorylase has been isolated and purified from Bacillus stearothermophilus, a thermophilic bacterium belonging to the genus Bacillus, and its enzymatic properties have been studied and reported (J. Biol. Chem .. 244, 3891). ~ 3697 (1969), Agric. Biol. Chera., 53, 2205-2210 (August 23, 1989),

Agric. Biol. Chem .. 53, 3219-3224 (December 23-1989))). In addition, a method for producing 5-methylperidine or thymidine using microbial cells of Noctilus stearothermophilus as an enzyme source has been reported (Japanese Patent Application Laid-Open No. 11-23095). No. Gazette (published on December 27, 1989), Agric. Biol. Chem,, 53. 197-202 (January 23, 1989)). However, although the nucleoside phosphorylase reported above has the advantage of having heat resistance, it has a low specific activity and a low enzyme activity per unit weight of bacterial cells, so that it is possible to produce nucleosides in good yield. It was not enough to solve the conventional problem of not being able to do so. That is, the nucleus described in Japanese Patent Application Laid-Open No. 1-32095 If the yield of the nucleoside is expressed as a ratio to the base donor used, at most around 30% (even if it is ideally reacted, the yield of the target product obtained from the equilibrium constant of the enzyme reaction is 53 to 5%). 6%).

 Disclosure of the invention

 The present inventors have found that a microorganism containing a large amount of thermostable nucleoside phosphorylase having high specific activity and high heat resistance, and having high nucleoside phosphorylase activity per unit weight of bacterial cells, as described above. As a result of further study on the group, ① These microorganisms have high specific activity, heat-resistant nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, and contain nucleosidase. Not exhibiting any very weak activity at the reaction temperature (35-80) during the production of nucleosides, even if it is contained, and The use of enzyme preparations containing nucleoside phosphorylase from the cells of one or more microorganisms for the enzymatic production of nucleosides requires only a small amount. And found that it is possible to produce a good yield quinuclidine Reoshi de in a short time the amount of enzyme and completed the present invention.

That is, in the present invention, a base donor, a sugar residue donor, and a phosphate donor are reacted using a nucleoside phosphorylase preparation, and the base moiety of the base donor and the sugar moiety of the sugar residue donor are reacted. An N-glycosidic bond is formed between In the method for producing a phosphide, a nucleoside phosphorylase-containing enzyme preparation is selected from the group consisting of one of thermophilic bacteria belonging to the genus Bacillus which has a high nucleoside phosphorylase activity per unit weight of the bacterial cell. The present invention relates to a method for producing a nucleoside, which is characterized by using a substance derived from the species of two or more microorganisms.

 As used herein, the term "nucleoside" includes naturally occurring nucleosides such as peridine, thymidine, cytidine, adenosine, and guanosine, as well as various nucleoside analogs. This indicates the range to perform. The present invention also relates to the above enzyme preparation itself, a novel microorganism used for preparing the enzyme preparation, and a novel nucleoside phosphorylase prepared from the microorganism and which can be used for production of nucleoside. Things.

 BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the optimum pH and stable pH range of the purine nucleoside phosphorylase of the present invention. FIG. 2 shows the optimum temperature and stable temperature range of the purine nucleoside phosphorylase of the present invention. FIG. 3 shows the optimum pH and the stable pH range of the pyrimidine nucleoside phosphorylase of the present invention. FIG. 4 shows the optimum temperature and the optimum temperature of the pyrimidine nucleoside phosphorylase of the present invention. Fig. 5 shows the stable temperature range. —2 and Brevibacterium acetylicum AT 6-7 were used as enzyme sources to produce 11-S-D-ribofuranosyl 1,2,4-triazole-3-carboxamide (rivapyrin). It is a comparison of rivapirin production rates at different reaction times.

 BEST MODE FOR CARRYING OUT THE INVENTION I. Enzyme Preparation Containing Nucleoside Phosphorylase The “enzyme preparation containing nucleoside phosphorylase” (hereinafter referred to as “enzyme preparation”) of the present invention includes: The term refers to those containing at least one of nucleoside phosphorylase or pyrimidine nucleoside phosphorylase, but preferably both nucleoside phosphorylases.

 The term “degree of purification of the enzyme” means the ratio of the amount of the enzyme protein to the total amount of the protein.

 The enzyme preparation of the present invention includes thermophiles belonging to the genus Bacillus, specifically, Bacillus It can be prepared from microorganisms belonging to a group of microorganisms having high nucleoside phosphorylase activity per unit weight of cells (hereinafter referred to as microorganisms of the present invention) among microorganisms belonging to moderately thermophilic bacteria such as stearotherniophilus).

Nucleoside phosphorylase for selecting microorganisms For the activity, for example, the following values are a rough guide.

 ♦ Prin nucleoside phosphorylase

 1 O UZg wet cells or more, preferably 12 U / g wet cells or more

 · Pyrimidine nucleoside phosphorylase

 1 wet cell or more, preferably 15 UZg wet cell or more

 Those satisfying one of these two conditions can be used as a preparation source of an enzyme preparation used in the present invention, and those satisfying both of the above two conditions at the same time It is suitable as a preparation source for products.

 Specific examples of suitable microorganisms satisfying such conditions include Bacillus stearothera (Philas TH62, P-21 and P-23) (both in the hamasa soy sauce premises) (Soil isolates isolated from soil). The bacteriological properties of the TH6-2 strain, which is the most representative strain of the strain group, are shown below.

 Mycological properties of H 6-2:

 (Α) Shape

 ① Cell shape and size

 Short rod (Rod), 0.6-: L.12-7111

② Spore presence Yes

 ③ Spore-sac swelling Yes

部位 Location and size of intracellular spores Terminal or central, 0.8 X 0.8-: L. 0 m ⑤ Gram stainability

 Gram variable (Gram positive at the beginning of culture)

 (B) Growth status in each medium

 ① Gravy agar slant medium

 Growth aspect: vigorous growth, smooth surface, opaque, no change in medium

 ② Gravy agar plate medium

 Growth aspect: Round colony formation, thin spread, viscous, opaque, fringing

 ③ Litmus · Milk medium

 Do not grow

 (C) Physiological properties

 ① Growing in the presence of oxygen: Growing

 ② Growth in the absence of oxygen: No growth

 ③ Catalase: positive

 ④ V—P test: negative

 メ チ ル Methyl red test (pH in V-P broth): <pH6

加 水 Hydrolytic capacity casein: negative

 Gelatin: Positive Starch: Positive

 ⑦ Use of citrate: positive

 硝酸 Nitrate reduction: positive

 ⑨ Indole formation: negative

ナ Sodium chloride or Requirement of chloride room: Negative

 酸 Acid production from carbohydrates

 Positives: glucose, arabinose, xylose, fructose, maltose

 Negative: starch, glycerol, sucrose, raffinose

 © Growth at each pH

 Growing at 6.8, not growing at 5.7

 生 Growth in the presence of sodium chloride

 In the presence of 2% Na C 1: grows

 In the presence of 5% Na C 1: Does not grow

 範 囲 Growth range

 Growth PH range: 6.5 to 9.0

 Growing temperature range: 35 to 65

 Growth in the presence of glucose

Glucose did not grow in the presence of 0.5% or more. When these mycological properties were checked against the classification criteria of Bergey's Manual of Systematic Bacteriology (8th edition), the isolated bacteria were Bacillus ♦ stearoser. It was found to belong to the morphophilus, and was named Bacillus ♦ stearoser morphiras TH 6-2. P-21 and P123 also showed the same mycological properties. TH-6-2 has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology under the Budapest Treaty. No. 2 758 is given. This international deposit was transferred on February 16, 1990 from No. 10526, deposited on the depository organization in Japan on February 4, 1989.

TH 6-2, P-21, and P-23 all belong to the Bacillus ♦ stearoser philus, but have a very high nucleoside phosphorylase activity per unit weight of bacterial cells and have a high level of nucleosidase activity. It is clearly distinguished from known microorganisms in that it does not substantially contain it. For example, using a known microorganism belonging to Bacillus stearothermophilus stored in the American ♦ Type ♦ Culture 'Collection (ATCC) and the above isolated bacteria, cell units can be used. Table 1 shows the results of a comparative study of the activity of bilimidine nucleoside phosphorylase and purine nucleoside phosphorylase per weight. From Table 1, it can be seen that the nucleoside phosphorylase activity per unit cell weight of D-H6-2, P-21 and P-23 is higher than that of the previously known strains. It can be seen that the activity is slightly less than 2-fold for nucleoside phosphorylase activity and slightly more than 6-fold for pyrimidine nucleoside phosphorylase activity. Specifically, the above isolated bacteria have a purine nucleoside phosphorylase activity and a pyrimidine nucleoside phosphorylase activity per unit cell weight of 13 to 15 U / g wet cells and 20 to 50 U / g, respectively. The value of 22 U / wet cells was shown. Table 1

Therefore, this isolated bacterium sufficiently satisfies the selection criteria for nucleoside phosphorylase activity described above, and is useful as a preparation source for an enzyme preparation used for producing nucleosides.

To prove this, we produced ribavirin and compared the production rate of the target compound with known strains. As a result, as shown in Table 2, when microorganisms belonging to the microorganism group of the present invention were used, all of them showed a production rate of 90% or more, while known microorganisms showed a production rate of at most 40%. Only the production rate was shown, and it was confirmed that the microorganism belonging to the microorganism group of the present invention is extremely useful as an enzyme source for producing nucleosides.

Table 2

Rinopyrine pirates (%) Ratio of microorganisms used to 1,2,4-triazole-3-carboxamide>

^ 9e ⁿ JimS.W 丄 Jl one b— 95.0

 "D one 1 u

 "CP—23) 94. 1

 • A i

 · O

 〃 "Q U Π Π U 30. 3

 〃 丄 u 丄 4 y 11.7

 // 1 丄 Π u 1 丄 o 44. 4

 〃 12976 8.0

 〃 12978 43.5

 〃 12980 0

 〃 15952 39.0

 〃 21365 37. 6

〃 29609 4.1 In the above comparative test, the cells of the microorganism were prepared in the same manner as in Example 1 described below, and the measurement of the activity of prinnucleoside phosphophorylase and pyrimidine nucleoside phosphorylase was performed using the titer described below. The measurement was performed according to the method described in In addition, livapirine is prepared by using a substrate solution (pH containing 40 mM 1,2,4-triazole-3-carboxamide, 60 mM resin, 40 mM dihydrogen phosphate). 6.0 aqueous solution) The suspension was prepared by adding 1 Oml (containing 200 nig as wet cells) of a cell suspension in which the cell weight was adjusted to 1 Oml, and stirring at 50 for 24 hours. After the above reaction, the reaction solution was centrifuged, and the supernatant was diluted 50- to 100-fold, and this was subjected to the HPLC method (column: YMC A

-3122 (manufactured by Yamamura Chemical Laboratory), eluent: 0.15M dihydrogen phosphate solution in water, detection: absorption at 220 nm), and the amount of rivapirine was measured.

 The generation rate was determined by the following equation. Concentration of generated ribavirin (M)

 Generation rate (¾) = X 100

Concentration of 1,2,4-triazole-3-carboxamide used (M) The enzyme preparation of the present invention is obtained by culturing a microorganism belonging to the microorganism group of the present invention and culturing the cells. Can be prepared by appropriately processing into a use form according to the purpose of use. A culture medium for culturing microorganisms contains an appropriate amount of a carbon source and a nitrogen source which can be assimilated by these microorganisms, and if necessary, a metal salt, a trace growth promoting substance, an antifoaming agent, etc. The added one is used. Specifically, medium components include sugars (glucose, saccharose, etc.), natural carbohydrates (molasses, molasses, starch, wheat, bran, rice, etc.), alcohols, fatty acids, hydrocarbons Nitrogen sources include meat extract, yeast extract, soy hydrolyzate, casamino acids, various amino acids, urea, etc.Inorganic salts include zinc, iron, magnesium, sodium, calcium, calcium Examples of trace growth-promoting substances, such as phosphates, hydrochlorides, and sulfates of metals such as chromium, include vitamin 8 2, vitamin B ₂ , amino, pentatothenic acid, and biotin.

 Culture may be performed by a conventional liquid culture method (eg, supernatant culture, aeration-agitation culture, stationary culture, or continuous culture).

 Culture conditions vary depending on the microorganism and the type of medium, and cannot be specified. Usually, the pH at the start of the culture is adjusted to 6.5 to 9.0, and the temperature is adjusted to about 35 to 65 ° C until the desired enzyme activity is sufficiently obtained. Incubate for about 50 hours.

The embodiment of the enzyme preparation prepared using the culture solution containing the viable cells thus obtained (hereinafter referred to as culture) is not particularly limited. For example, the culture of the microorganism itself, culture Normal separation means (centrifugation, sediment -1 1-

(Separation, coagulation separation, washing, etc.) can be exemplified.

 More specific examples of processed cells of live cells are mechanical destruction of live cells (using a waling ♦ blender, French ♦ press, homogenizer, mortar, etc.), freeze-thawing, drying (freezing) Drying, air drying, acetate drying, etc.), autolysis (using a solvent such as toluene or ethyl acetate), enzyme treatment (using cell wall lysing enzymes such as lysozyme), ultrasonic treatment, osmotic pressure treatment Destruction of viable cells obtained by treatment in accordance with general treatment methods such as chemical methods, chemical treatment (using salt solution, acidic solution, alkaline solution, surfactant, chelating agent, etc.) Denature the cell wall or Z of living cells and the cell membrane, or extract a fraction with enzymatic activity, and if necessary, extract the extract with enzymatic activity into a common enzyme. Purification method (By salting-out treatment, isoelectric point precipitation treatment, organic solvent precipitation treatment, various chromatographic treatments, dialysis treatment, etc.) to fractionate the fraction having the enzyme activity desired by the present invention. And a crude enzyme obtained by the above method.

 Such cultures, viable cells and treated cells may be used in the free state without immobilization treatment, and may be immobilized by ordinary methods such as inclusive method, cross-linking method and adsorption method. It may be used as an immobilized material.

Regarding the purified yeast cord which is one aspect of the processed bacterial cells, To be more specific, the nucleoside phosphorylase obtained by extraction and purification from Bacillus stearothermophilus TH6-2 belonging to the microorganism group of the present invention has the following enzymatic properties.

 (A) Prin nucleoside phosphorylase

 (1) Operation

 Prin nucleoside + phosphoric acid

 ^ Purine base + pentose-1-phosphoric acid

 The purine nucleoside phosphorylase of the present invention catalyzes the above-mentioned phosphorylation reaction. For this reason, it belongs to E. C. 2.4.2, 1 of the International Enzyme Classification.

 (2) Substrate specificity

Table 3 shows the results of phosphorylation of various purine nucleotides as substrates.

9

Table 3

Base Amount of base generated Relative activity

 (n inol / lOfliin) (X) f D J 0.1

2-"Sai-chan"

3-: r-year-old: r-no-shin 0 0 no-no-no-non-no-

 0 0 Innosin 2.5 5 1 0 0

2'-doxyinosine 2.54 1 0 0

3'-Doxy-no-syn 0 0 Guan-o-sin 2. 1 5 84

2'-Doxyguanosine 2.1.8 8 5

From Table 3, it can be seen that the purine nucleoside phosphorylase of the present invention is specific for inosine, 2'-deoxyinosine, guanosine and 2'-deoxyguanosine within the range tested.

(3) Optimal pH and pH stability

 The optimal pH is pH 7-8, and the stable pH range is pH 5-9 (see Figure 1).

 (4) Optimum temperature and temperature stability

 The optimum temperature is 60-80 ° C: the stable temperature range is up to 60 (see Fig. 2).

 (5) Molecular weight

 The molecular weight is about 45,000 as measured by SDS-Polyacrylamide gel electrophoresis. '

 (6) Titer (specific activity)

 The specific activity is more than 400 (U / mg) at 80% enzyme purification and 450 (U / mg) at 90% enzyme purification.

 (B) Pyrimidine nucleoside phosphorylase

 (1) Operation

 Pyrimidine nucleoside + phosphoric acid

^ Pyrimidine base '+ pentose-1-phosphoric acid The pyrimidine nucleoside phosphorylase of the present invention catalyzes the above-mentioned phosphorylation reaction. For this reason, it belongs to the International Enzyme Classification EC 2.4.2.2. (2) Substrate specificity

 Table 4 shows the results of phosphorylation of various pyrimidine nucleotides as substrates.

 Table 4

Base production relative activity ((1 raol / lOniin) (X)) resin 1.73 68

2'-Doxy resin 2.55 1 00 ARA BINOFURANO CYLINDER 0 0 LUCY DOU LIZIN 0-0 CYCLOW LIZIN 0 0 CITIDINE 0 0 1

2'-Doxycytidine 0 0 Ribofuranosyl thymine 0.96 38 Thymidine 1.7 1 67

As shown in Table 4, the pyrimidine nucleoside phosphorylase of the present invention is specific to peridine, 2'-dexoxylysine, ribofuranosylthymine, and thymidine within the range tested.

 (3) Optimal pH and pH stability

The optimal pH is pH 7-9, and the stable pH range is pH 5-9 (see Figure 3). (4) Optimum temperature and temperature stability

 The optimum temperature is 60 to 70, and the stable temperature range is up to 60 ° C (see Fig. 4).

 (5) Molecular weight

 The molecular weight measured by SDS-polyacrylamide electrophoresis is about 31,000.

 (6) Titer (specific activity)

 The specific activity is more than 250 (U / ing) at 80% enzyme purification and 297 (U / mg) at 90% enzyme purification.

 The above enzymatic properties were measured by the following methods. ① Measurement of titer,

Prin nucleoside phosphorylase activity-Substrate solution (pH 8.0 aqueous solution containing 20 mM inosine and 0.1 M potassium dihydrogen phosphate) 1.0 ml of enzyme solution (1 g as purified enzyme) containing 50 mM齚酸after buffer (p H 6. 0)) 20 £ added and reacted for 10 minutes at 50 ^e C, reaction by adding hydrochloric acid so that such a 0. iN at a final concentration Stop and cool at 0 for 10 minutes. Next, the reaction solution was centrifuged, and the obtained supernatant was subjected to an HPLC method (column: YMC A- 312 (manufactured by Yamamura Chemical Laboratory), eluent: 20 mM toluene containing 5.0% of acetonitrile). Quantify the amount of hypoxanthine produced in squirrel-HCl buffer (pH 7.5), detection: 260 nm). In one minute One two

1 unit of enzyme producing 1 μmol hypoxanthine

(ru)).

 Pyrimidine nucleoside phosphorylase activity

 The procedure is the same as the above-described method for measuring the phosphoryl phosphorylase activity except that peridine is used instead of inosine in the substrate solution and peracyl is quantified by the HPLC method. One unit is the amount of enzyme that produces 1 mol of peracil per minute.

 ② Substrate specificity

 Using an aqueous solution of pH 8.0 containing 10 mM of various nucleosides and 50 mM of potassium dihydrogen phosphate as a substrate solution, react at 50 ° C for 10 minutes. The procedure is the same as that for the measurement of the activity of purine nucleoside phosphorylase, except that the bases of various nucleosides are quantified by the method.

 ③ Optimal pH

 Dissolve nucleosides (20 mM inosine or peridine) and 0.1 M potassium dihydrogen phosphate, and adjust the pH to 4 with an aqueous solution of dilute hydrochloric acid or dilute sodium hydroxide. The procedure is performed in the same manner as in the measurement of the activity of purine nucleotide phosphorylase, except that the substrate solution adjusted to ~ 10 is used.

安定 Stable P H

Store in 0.2M acetate buffer (pH 3.5-6) and Tris-HCl buffer (pH 7-9) at 37 for 16 hours The procedure is the same as that for measuring the activity of purine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase, except that the enzyme solution used is used.

 適 Optimal temperature

 The reaction is performed in the same manner as in the measurement of the activity of purine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase, except that the reaction is carried out at each temperature of 30 to 80.

⑥ Stable temperature range

 Except for using an enzyme solution heated at 30 to 80 for 15 minutes, the measurement is performed in the same manner as in the method for measuring the activity of purine nucleoside phosphorylase or pyrimidine nucleoside phosphorylase.

 The characteristics of the enzymes obtained from the microorganisms in the microorganism group of the present invention described above are that they have an optimum temperature and a stable temperature range to a relatively high temperature, and have a very high specific activity. Therefore, if a preparation containing an enzyme having such characteristics is used for the production of a nucleotide, the amount of the enzyme preparation used in the reaction can be reduced, and the nucleotide amount can be reduced with a small amount of the enzyme. Can be manufactured with good yield. Furthermore, since the reaction can be performed at a relatively high temperature (45 or more), contamination by various microorganisms can be prevented.

 製造 Manufacture of nucleotides

The production of a nucleoside using the above enzyme preparation is carried out in a reaction vessel in the form of a base donor, a sugar residue donor and a sugar residue donor described below. The reaction can be carried out by contacting a phosphoric acid donor with an enzyme preparation.

① Base donor

 The base donor used in the method of the present invention supplies a base to the reaction system. The base donor to be used may be selected according to the target nucleotide, and the N-glycosidic bond between the sugar moiety of the sugar residue donor and the N-glycosidic bond is formed by the action of nucleoside phosphorylase. Examples of the heterocyclic base to be formed or a derivative thereof can be given. Specific examples of the heterocyclic base include, for example, purine and its derivatives, pyrimidine and its derivatives, triazole and its derivatives, imidazole and its derivatives, azapurine and its derivatives, azapurine and its derivatives, And azapyrimidine and its derivatives or pyridine and its derivatives. In addition, the base donor may be not only the heterocyclic base itself, but also a nucleotide or a nucleotide having the heterocyclic base.

Specifically, a substituent (for example, an amino group, a substituted amino group, a hydroxyl group, an oxo group, a mercapto group) is provided at one or more positions of the 1, 2, 6, or 8 positions of the prim base. Derivatives having a group, an acyl group, an alkyl group, a substituted alkyl group, an alkoxyl group, a halogeno atom), for example, adenine, guanine, hypoxanthine, xanthine, 6-mercaptoprin, 6-thioguanine, Ν ^σ -alkyl Or acyladenine, 2-alkoxyadenine, 2-thioadenine, 2,6-diaminoprin, etc .; at one or more of the 2-, 4- or 5-positions of pyrimidine, the same as above Pyrimidine derivatives having a substituent, for example, cytosine, peracyl, thymine, 5-no, logenouracil (eg, 5-fluorouracil, 5-hydroxyuracil, etc.), 5-halogenosit Shin (5 - Furuoroshi Toma, etc.), 5 - Application Benefits halogenoalkyl methyl © La sill (5 - Application Benefits Furuorome Chiruura sills etc.), 2 - Chioshi Toma, 4 - Chioura sills, N ⁴ - Ashirushi Toma, 5 - 1,2,4-triazole derivatives having a substituent at the 3-position of 1,2,4-triazole, for example, 1,2,4-triazole-3-carboxamide, 1,2,4-triazole derivative and the like; , 2, 4-G Azole-3-carboxylic acid, 1,2,4-triazole-3-carboxylic acid alkyl ester, etc .; imidazole derivative having substituents at 4- and 5-positions of imidazole For example, 5-amino-4-imidazolecarboxamide, 4-carbamoinole-imidazole-5-oleate, benzimidazole, etc .; Or a azapurine derivative at the 7-position, e.g., 1- azaadenine, 3- azaadenine, 3- azaguanine, 7- azaadenine, 7- azaguanine, or a substituent similar to the above pyridine derivative. 8-azaazadenine, 7-aza-8-azahipoxa Derivatives such as azapurinol (aroprinol); azapyrimidine derivatives such as 5-azathymine, 5-azacitosine, 6-azaduracil; and azapyrimidine derivatives such as 3-dezaduracil, nicotinic acid, and nicotinic acid amide Examples include pyridin derivatives.

 ② Sugar residue donor

 The sugar residue donor supplies a sugar residue to the reaction system. In other words, the sugar residue donor may be selected according to the target nucleotide, and an N-glycosidic bond is formed with the base moiety of the base donor by the action of nucleoside phosphorylase. For example, a ribose compound and a deoxyribose compound can be exemplified. Examples of ribose compounds include ribonucleosides such as inosine, guanosine, peridine, ribofuranosylthymine, and ribose-1-phosphate, and examples of deoxyribose compounds include: 2'-deoxyinosine, 2'-deoxyguanosine, 2'-deoxyperidine, thymidine, 2 ', 3'-dideoxyinosine, 2', 3'-dideoxyguanosine, 2 ', 3' -Doxy nucleosides such as didoxyperidine, 3'-deoxythymidine, and 2-dexoxyribose-1-linoleic acid, and 2,3-dideoxyribose-1-linoleic acid. it can.

When an enzyme preparation other than a purified enzyme is used, in addition to the above sugar residue donor, an additional enzyme may be used. Ribose compounds such as nosin, cytidine and xanthosine, and deoxyribose compounds such as 2'-deoxyadenosine, 2'-dexoxytidine and 2'-dexoxyxanthosine have the potential to be used.

③ Phosphoric acid donor

 As the phosphate donor, any one capable of dissociating into phosphate ions in the reaction solution may be used. For example, free phosphate or phosphate (eg, sodium, potassium) Alkali metal salts, ammonium salts, etc.) are preferably used. As the phosphate donor, a system capable of releasing phosphate ions in the reaction solution, such as a combination of various phosphate ester derivatives and phosphatase, or a combination of nucleotide and nucleotidase is used. You can also. ④ Reaction conditions

 As the reaction solution, a solution prepared by dissolving or suspending a base donor, a sugar residue donor and a phosphate donor in water or a buffer solution is used. The nucleoside corresponding to the base donor is enzymatically produced.

The concentrations of the base donor, sugar residue donor and phosphoric acid donor used may be appropriately selected from the range of 0.1 to 500 mM. The reaction usually proceeds efficiently in the range of 35 to 8 CTC, but a reaction temperature of about 40 to 70 is particularly preferred. When the reaction temperature is 35 or less, the reaction speed is slow and the reaction efficiency is low. not good. At a reaction temperature of 80 ° C or more, nucleoside phosphorylase activity may be reduced.

 The pH of the reaction solution is usually maintained in the range of pH 5 to 10, preferably pH 5 to 9. When the pH fluctuates during the reaction, the pH may be adjusted to a preferable pH range using an acid or alkali.

 After the reaction, the reaction solution and the enzyme composition are separated, and the target nucleoside is subjected to a separation and purification step.

 The generated nucleoside can be separated and purified by a known method or a method using the same. For example, use of various liquid chromatography such as ion exchange chromatography, adsorption chromatography, distribution chromatography, gel filtration, counter-current distribution, and counter-current extraction. Common separation and purification methods used in the separation and purification of nucleotides, such as methods that utilize differences in solubility such as concentration, cooling, and addition of organic solvents, etc., alone or in combination as appropriate. Just do it.

 〔Example〕

 Hereinafter, the present invention will be described specifically with reference to Examples and Comparative Examples.

Example 1

Sterile extract containing 0.5% Suemo extract (Difco), 1.0% peptone (Dco), 0.7% meat extract (Difco) and 0.3% salt Medium (pH 7.0) Bacillus ♦ stearothermophilus TH 6-2 (No. 2758, No. 2758) was inoculated into 500 ml, and cultured with shaking at 5 TC for 18 hours.

 The obtained culture was centrifuged to collect the cells, and after washing, sterilized water was added to prepare a 250-ml cell suspension. A 10 ml aliquot of this bacterial cell suspension was taken, and a substrate solution (pH 6.6) containing 40 mM base donor, 40 mM sugar residue donor and 40 mM dihydrogen phosphate reagent was prepared. 0) 10 ml was added, and the mixture was reacted at 40 to 60 with stirring.

 After the reaction, the amount of various nucleotides produced was determined by high performance liquid chromatography (column: YMC A-312 (manufactured by Yamamura Chemical Laboratory), eluent: 2.5 to 5% acetonitrile The measurement was carried out using a 2 OmM Tris-HCl buffer containing ril (pH 7.5), detection: absorption at 250 to 260 nm. The nucleoside generation rate was determined by the following equation.

Concentration of generated nucleoside (M) Production rate) X 100 Concentration of base donor used (M) The results are shown in Table 5. Table 5

 * ± t / It iH. (± product reaction condition generation rate (%)

• uni ^ l レ ^ ί ^ Ί * υ ^ ο- レ 、 rr

 Temperature CO time (hr) <to salt thigh> adenine ゥ resin adenosine 40 24 61

// 50 2 45

// 〃 〃 50 24 82

〃 〃 60 2 60

 〃 〃 60 24 85 Adenine 2'-deoxyxidiridine 2'-Doxyadenosine 40 2 25

〃 〃 〃 50 2 55

〃 〃 60 2 63

〃 〃 〃 60 4 70

〃 〃 〃 60 8 78 、,

// 2 ', 3'-dideoxyxidyridine 2 * .3'-dideoxyadenosine 55 24 15 ¹ ) thymidine peridine ribofuranosyl thymine 40 2 48

〃 // 〃 50 2 58

〃 // 〃 60 2 60

 〃 〃 60 24 65 Thymin 2 '

〃 // 〃 50 2 55

// // 〃 60 _ί 2 66

// // 〃 60 8 67

1) Shown in the ratio of sugar to donor.

Example 2

 Performed using a substrate solution (pH 6.0) containing 20 mM aloprinol as a base donor, 30 mM peridine and 75 mM dihydrogen phosphate as a sugar residue donor. The reaction was carried out at 60 ° C for 8 hours in the same manner as in Example 1 to produce a ribofuranosyl form of alprinol with a yield of 95% (ratio to alprinol).

Example 3

Base donors (aloprinol, benzimidazole, 6-mercaptoprin, purine, 6-thioguanine) 20 mM. Sugar residue donors (peridine, 2'-dexoxylysine) 30 mM and The reaction was carried out at 50 ° C for 8 hours in the same manner as in Example 1 using lithium dihydrogenate (30 mM), and the ribofuranosyl compound or 2'-deuterine having various base donors as bases was reacted. An oxyribofuranosyl compound was produced, and the results are shown in Table 6.

Table 6 Production rate of salt donor (%) Sugar residue donor

 <Base ratio> aloprinol peridine 80

 〃 2 '-Deoki 97

 Original benzimidazolidin 75

 2 '-Deoki 86 Series

 6-Mercaptoprin resin 50

 〃 2 '-Deoki 43

 Series

 Prince Resin 94

 〃 2 '-Deoki 65

 Series

 6-Choguanine resin 9

 〃 2 '-Deoki 65

 Series

Example 4

 40 mM 1,2,4-triazole-3-carboxamide (hereinafter referred to as “triazole”) as a base donor, and 40 mM pyridine and inosine as sugar residue donors , Cytidine, adenosine or guanosine,

Substrate solution containing 40 mM lithium dihydrogen phosphate

(pH 6.0) in the same manner as in Example 1. 4 one

Manufactured. The results are shown in Table 7,

Reaction conditions Production rate (%) Sugar residue donor Temperature time (vs. triazole

 (° C) (hr) ratio)

 ゥ Resin 60 4 94.2 Inosine 67 48 83.6 Cytidine 50 24 92.9 Adenosine 67 24 72.7 Guanosine 67 24 98.4 The nucleoside phosphorylase of the present invention is used for cytidine and adenosine. Since zeotide does not recognize it as a substrate, it is considered that it is used as a substrate after being converted to peridine and inosine by coexisting deaminase, respectively.

Example 5

A substrate solution (pH 6.0) containing 40 mM triazole as a base donor, 60 mM inosine as a sugar residue donor, and 40 mM rhodium dihydrogen phosphate was used. Similarly, the reaction was carried out at each reaction-temperature (40 to 70 ° C) for 24 hours to produce ribavirin. Table 8 shows the results. 5 one

Table 8

Reaction temperature 40 50 60 63 65 67 70

(° C) Production rate (! ¾)

 (To 37.0 58.0 71.7 73.7 76.0 78.7 77.9 sol ratio)

Example 6

 The culture obtained by culturing in the same manner as in Example 1 was centrifuged to obtain viable cells. Next, the viable cell 2. was suspended in 0.1 ml of 0.1 M Tris-HCl buffer (pH 7.0), and the light-curable resin (ENT-2000; Kansai Paint II) prepared separately was suspended in Oml. The above cell suspension was added to a resin solution in which 8.0 g of benzoethyl ether as a photopolymerization initiator was added to 8.0 g, mixed well, and then poured onto a transparent film. Then, a light beam of about 360 nm was simultaneously irradiated on the front and back of the film surface for 3 minutes to obtain a photopolymer. A portion containing 0.2 g as a cell mass is taken from the immobilized product, cut into small pieces, and then cut into 40 mM triazole, 40 mM lysine and 60 mM dihydrogen phosphate. The above immobilized product was added to 1 Oml of a substrate solution (pH 6.0) containing a solution, and stirred at 6 ° for 8 hours to produce rivapirin.

As a result of measuring the production rate by the aforementioned HPLC method, the production rate of rivapirine was 90% in terms of triazole ratio. Further, this reaction was performed 10 times continuously, but the production rate of ribavirin was maintained at 90%, and a decrease in enzyme activity was observed.

Example 7

 Triazole, peridine or inosine, and dihydrogen phosphate rim were added to the culture obtained in the same manner as in Example 1 to a final concentration of 40 mM, respectively. Shaking culture was performed at 0 ° C (when using peridine) or 65 ° C (when using inosine) for an additional 24 hours. After cultivation, the centrifugal supernatant was used to measure the production rate of ribavirin by the HPLC method. As a result, the production rate of ribavirin using lysine was 91.9% in triazole ratio, and When used, the yield was 65, 9%.

Example 8

 Preparation of purified enzyme

Bacillus stearotherer 乇 TH6-2 from bouillonland containing 1.0% peptone, 0.4% broth, 0.5% yeast extract, 0.3% salt, pH 7. The cells were inoculated into a large test tube containing 1 Oml of the medium adjusted to 2, and cultured at 50 overnight. The obtained culture was transferred to a 300-ml flask containing 30 ml of a medium having the same composition and the same pH, and cultured at 50 for 8 hours. 5 pound jar including ♦ Fermenter, 50, rpm 3500 rpi, air flow rate 1. O vvn Culture for 18 hours. Approximately 30 g of wet cells were obtained from the thus obtained culture solution by centrifugation, and 1.5% of 0.1% Triton X-100 (manufactured by Shidama) was obtained.

The suspension was suspended in 50 mM Tris-HCl buffer (pH 7.2) containing 5 mM EDTA, and 75 mg of lysozyme (manufactured by Sigma) was added, and the mixture was kept at 37 for 1 hour. The lysate was centrifuged at 8,000 rpm to remove cell debris, adjusted to pH 6.0 by adding 2N hydrochloric acid, and heat-treated at 50 for 5 minutes. And centrifuged to obtain a supernatant as a crude enzyme solution.

The crude enzyme solution was fractionated by salting out using ammonium sulfate, and the protein precipitate obtained at 40% to 90% saturation was added to a 50 mM acetic acid-sodium acetate buffer (pH 6.0). lysed, overnight dialyzed against large volumes of the same buffer, the resulting inner solution centrifuged _t supernatant above acetate buffer to remove the precipitate formed during dialyzed with (hereinafter buffer a and Column) equilibrated in the above)

(2.2 x 60 cm), and the adsorbed protein was eluted with a linear gradient method using 0-0.5 M salt (using buffer A), and the purine nucleoside phosphorylase fraction and pyrimidine nucleoside were eluted. Each of the dophosphorylase fractions was collected. After dialysis of each active fraction against buffer A, the same operation as above was performed using 2 Oml DEAE Toyopearl resin filled in a 25 ml syringe (manufactured by Terumo Corporation). -3 S-

Column chromatography was performed to collect each active fraction. Next, these active fractions were gel-filtered using a Toyopearl HW-55S (Tosoh Corporation) column (2, 4 x 80 cm) equilibrated with buffer 1A, and the components were leached. Was recovered as a substantially uniform purified sample.

 Table 9 shows the amount of protein, the total activity, and the degree of purification of the active fractions in the purification process of both enzymes.

The degree of purification was determined by measuring the relative ratio of each band using a densitometer on the electrophoretic pattern obtained by SDS-polyacrylamide gel electrophoresis.

笫 9 Table

Φ¾white S is 608 Omg. »The amount of protein is 377 Omg <

Comparative Example 1

 Brevipacterimu ♦ acetylicum AT 6-7 (ATCC 3931 1 1) and Bacillus ♦ stearoza monomorphus TH 6-2 The culture medium (peptone 1%, meat extract 0.7%) A large test tube containing 0.3%, 0.3% of salt, 0.5% of yeast extract, and 10 ml of pH 7.2) was inoculated, and each was 28. The cells were cultured with shaking at C (for AT 6-7) and 50 ° C (for TH 6-2) for 18 hours. After the culture, each culture was centrifuged to separate the cells. After washing, cells of the same wet weight were suspended in 10 ml of deionized water.

 A substrate solution containing 0.4 mM triazole, 0.4 mM lysine and 0.4 mM dihydrogen phosphate was added to each of the cell suspensions (AT 6-7). If the pH is 7.◦ or TH6—2, adjust the pH to 6.) Add 10 ml and react with stirring at 45 ° C or 65 ° C in a closed system. The sample was sampled and the production rate of rivapirine was measured by the aforementioned HPLC method.

Fig. 5 shows the results. As is evident from Fig. 5, when the enzyme preparation prepared from the microorganism of the present invention was used, the target compound was produced in a shorter time than AT 6-7, which was conventionally a very excellent source of enzyme preparation. It was clarified that the enzyme preparation could be used in a small amount. As described above, the enzyme preparation containing nucleoside phosphorylase of the present invention contains a large amount of nucleoside phosphorylase having high specific activity and heat resistance, and contains a large amount of nucleoside phosphorylase per unit weight of bacterial cell. It is derived from one or two or more microorganisms belonging to the thermophilic bacterium belonging to the genus Bacillus, which has a high enzyme activity.These enzyme preparations are used for the production of nucleosides. It is a very useful and practical method that has the following features.

 (1) It contains a large amount of nucleoside phosphorylase with high specific activity. When used for nucleoside production, nucleoside can be produced with high yield with a small amount of enzyme.

 ② Contains nucleoside phosphorylase whose optimum and stable temperature ranges are relatively high. For this reason, the reaction can be performed at a high temperature, and the inactivation of the enzyme due to contamination of various bacteria and the decomposition of the reaction product can be suppressed.

③ It is also possible to obtain a preparation containing both of the enzymes, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, as nucleoside phosphorylases. For example, as shown in the following reaction formula, the two enzymes work together, and are twice as large as an enzyme preparation containing only one type of nucleoside phosphorylase. Nucleosides can be produced at the above speed. (Prin Nuku Reoshi

 Dophosphorylase)

 1

Sugar residue donor-> Sugar residue donor

 (Sugar portion of prin nu

 Leoside)

 Purine base

(Pirimi Jinnuk Leo

Side phosphorylase)

 1

 -Pyrimidine nucleoside individual--: Limidine base The microorganism of the present invention grows at a relatively high temperature and grows at a high growth rate. It is extremely useful as an enzyme preparation or a preparation source for use in nucleoside production because it contains a large amount of an enzyme suitable for the production of a nucleoside.

 Furthermore, when a culture of a microorganism is used as the enzyme preparation of the present invention, autolysis of the cells can be prevented.

In addition, the nucleoside phosphorylase obtained from the microorganism of the present invention has a high specific activity, and has an optimum temperature and a stable temperature range in a relatively high temperature range. They are clearly distinguished. Further, when such an enzyme is used for producing a nucleotide, the above-mentioned effects (1) and (2) are obtained. In addition, the purine nucleoside phosphorylase of the present invention is If both enzymes are used, naturally the above-mentioned effect (3) will be achieved.

 Industrial applicability

 The method for producing a nucleoside of the present invention comprises a large amount of heat-resistant nucleoside phosphorylase belonging to a thermophilic bacterium belonging to the genus Bacillus, which contains a large amount of nucleoside phosphorylase. Enzyme preparations derived from microbial cells of microorganisms with high enzyme activity are used as an enzyme source for the production of nucleosides, and the target nucleosides can be produced with a small amount of enzyme in high yield. This is a very practical method.

Claims

The scope of the claims 1. A base donor, a sugar residue donor, and a phosphate donor are reacted using an enzyme preparation containing nucleoside phosphorylase (se), and the base moiety of the base donor is reacted. A method for producing a nucleoside by forming an N-glycosidic bond between a nucleoside phosphorylase and a saccharide moiety of a bacterium belonging to the genus Bacillus. A method for producing a nucleoside, comprising using a microorganism derived from one or more microorganisms of a microorganism group belonging to a microorganism and having a high nucleoside phosphorylase activity per unit weight of the microorganism.  2. The enzyme preparation according to claim 1, wherein the enzyme preparation is a cell derived from a cell having a nucleotide phosphorylase activity per unit weight of the cell that satisfies at least one of the following conditions. Reoside manufacturing method.  ① Prin nucleoside phosphorylase activity  10 U / g wet cells or more  ② Pyrimidine nucleoside phosphorylase activity  1 O U / g wet cells or more 3. The enzyme preparation according to claim 1, wherein the enzyme preparation has a nucleoside phosphorylase activity per unit weight of the bacterium that satisfies the following conditions simultaneously. Manufacturing method. ① Prin nucleoside phosphorylase activity  10 UZg wet cells or more  ② Pyrimidine nucleoside phosphorylase activity  10 UZg wet cells or more  4. The method for producing a nucleotide according to any one of claims 1 to 3, wherein an enzyme preparation derived from a medium thermophilic bacterium of the genus Bacillus is used.  5. The method for producing a nucleotide according to any one of claims 1 to 3, wherein an enzyme preparation derived from Bacillus ♦ stearothermophilus cells is used.  6. The method for producing a nucleoside according to any one of claims 1 to 3, wherein the form of the enzyme preparation is a culture, a viable cell, or a processed cell of a microorganism.  7. A microorganism having a nucleoside phosphorylase activity per unit weight of the cell that satisfies at least one of the following conditions. ① Prin nucleoside phosphorylase activity  10 UZ g or more wet cells  ② Pyrimidine nucleoside phosphorylase activity  10 U / g wet cells or more  8. A microorganism whose nucleoside phosphorylase activity per unit cell weight satisfies the following conditions at the same time.  ① Prin nucleoside phosphorylase activity  10 UZ g or more wet cells ② Pyrimidine nucleoside phosphorylase activity More than 10 UZ g wet cells  9. The microorganism according to claim 7 or 8, wherein the microorganism belongs to a thermophilic bacterium of the genus Bacillus.  10. The microorganism according to claim 7, wherein the microorganism belongs to a medium thermophilic bacterium of the genus Bacillus.  1 1. The microorganism according to claim 7 or 8, wherein the microorganism belongs to Bacillus, Stearorisa filasus. 1 2. Purine nucleoside phosphorylase having the following properties.  ① Temperature stability: Stable even when heated for 60 to 15 minutes  ② Specific activity: 400 at 80% enzyme purification  (U / mg) or more  1 3. A pyrimidine nucleoside phosphorylase having the following properties.  ① Temperature stability: Stable even after heating for 60 or 15 minutes  ② Specific activity: 250% with 80% enzyme purification  (U / mg) or more